The prevalence of osteoarthritis (OA) and chondrocalcinosis (CC) increase with age and these degenerative cartilage disorders impose enormous societal costs. Current treatments have been primarily directed towards relief of symptoms and functional limitations, but do not tackle the root causes of OA and CC or prevent progressive deterioration of the joints. Our objective is to identify better therapeutic agents that preserve cartilage structure and function. MitoKor scientists, in collaboration with Dr. Terkeltaub at UCSD, have tested the hypothesis that dysregulated mitochondrial function plays a central role in pathogenesis of OA and CC. The effects of nitric oxide (NO), and IL- 1 are known to modulate the pathogenesis of OA and CC. Our studies have shown that (a) donors of NO and peroxynitrite inhibit mitochondrial respiration and ATP synthesis in cultured articular chondrocytes, and (b) direct inhibition of oxidative phosphorylation recapitulates key phenotypic features of the OA and CC chondrocytes. Subsequent studies have focused on identifying agents that attenuate the effects of specific pro-OA and pro-CC "triggers" (NO, peroxynitrite and IL- 1) on chondrocytes. Promising small molecule leads have been identified from cell-based screening assays that prevent NO release, matrix degradation and calcification in primary chondrocytes and in cartilage organ cultures. Importantly, the lead compounds effectively block trigger-induced ATP depletion and inhibition of intact cell respiration. Specific Aim 1 will test whether the efficacy of specific lead compounds is primarily due to direct preservation of mitochondrial function. Using permeabilized bovine chondrocytes, we will ascertain if the leads directly protect from trigger-induced respiratory inhibition (and so identify locus of protection). Specific Aim 2 will capitalize on the results of Aim 1. We will either (a) test for potential pathways that lie upstream of mitochondria in which the compounds may be exerting a protective effect, and will focus specifically on sphingomyelinase/ceramide signaling or (b) will probe specific deleterious mitochondrial events that may be inhibited by the compounds. These will include the mitochondrial permeability transition, cytochrome c release and mitochondrial glutathione depletion. These studies will help guide the identification of the signaling pathways involving mitochondria and to specific molecular targets that can be exploited for optimizing our therapeutic strategy for OA and CC.